A method for quantitatively detecting total phosphates in aquatic products by acid hydrolysis combined with liquid chromatography tandem mass spectrometry

Abstract

This invention relates to the field of analytical chemistry, and in particular to a method for the quantitative determination of total phosphate in aquatic products using acid hydrolysis combined with liquid chromatography-tandem mass spectrometry. The method exhibits good linearity for orthophosphate in the range of 0.1–20.0 mg / L, with a correlation coefficient of 0.9991, a limit of detection of 0.03 g / kg, a limit of quantitation of 0.10 g / kg, and low matrix effect. At spiking levels of 1.0–10.0 g / kg, the intra-day recoveries for shrimp and fish are 89.8%–102.8% and 88.7%–105.8%, respectively; the inter-day recoveries are 90.9%–106.4% and 90.2%–105.8%, respectively; the intra-day RSD is ≤10.0%, and the inter-day RSD is ≤9.6%. This method features simple pretreatment, strong anti-interference capabilities, high sensitivity, and excellent accuracy and precision. It is suitable for the detection of total phosphate in aquatic products, providing a reliable technical means for the supervision of total phosphate in aquatic products, and is highly practical.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a method for the quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Polyphosphates possess properties such as moisture retention, pH regulation, and improved taste, and are widely used as food additives in the processing and preservation of aquatic products. Commonly used polyphosphate water-retaining agents in aquatic products include pyrophosphates, tripolyphosphates, trimetaphosphates, and hexametaphosphates, used either individually or in combination. However, excessive use of polyphosphate water-retaining agents can lead to excessively high phosphate concentrations in the body, disrupting the calcium-phosphorus balance and potentially causing developmental delays, osteoporosis, urinary tract stones, and exacerbating cardiovascular diseases such as hypertension and heart disease, thus posing a threat to human health.

[0003] To effectively regulate the excessive use of phosphate water-retaining agents, the national food safety standard (GB 2760-2024) has imposed strict limits on the amount of phosphate used in aquatic products. Currently, the main methods for determining phosphate in aquatic products include spectrophotometry, near-infrared spectroscopy, and ion chromatography. Spectrophotometry, as a classic method for phosphate analysis, has drawbacks such as relatively cumbersome and time-consuming sample pretreatment, the toxicity of some reducing agents, and relatively low sensitivity. While near-infrared spectroscopy is a non-destructive method, the infrared spectral signal response is easily affected by factors such as the type of sample and moisture content, and its quantitative accuracy needs improvement. Ion chromatography is currently the most widely used method, with the advantage of being able to determine the composition and content of various phosphates in aquatic products. However, it also has several problems: firstly, pretreatment for ion chromatography often requires purification methods such as solid-phase extraction, increasing the complexity of pretreatment and detection costs; secondly, achieving the separation of multiple phosphates results in long chromatographic analysis times and low analytical efficiency. Furthermore, phosphate water-retaining agents are often used in the form of complex phosphates, which may contain multiple phosphates. The test results are generally calculated as total phosphate. Literature reports that polyphosphates in aquatic products are prone to degradation during thawing, extraction, and other processing, especially hexametaphosphate, which is highly decomposed. Therefore, determining the total phosphate content by separately quantifying each polyphosphate component using ion chromatography and then summing the results presents certain accuracy issues.

[0004] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is currently an ideal analytical method for small molecule compounds, offering advantages such as high detection sensitivity and strong resistance to matrix interference. Phosphates, as highly polar compounds, require specific chromatographic columns and are generally not retained on conventional reversed-phase columns. The limited availability of ideal columns may explain the scarcity of reports on the application of LC-MS / MS in the analysis and detection of phosphates.

[0005] Therefore, in order to improve the sensitivity, accuracy and analytical efficiency of phosphate detection in aquatic products, and at the same time meet the practical application requirements of food safety supervision for the specificity and convenience of detection methods, developing a new, efficient method for total phosphate detection that is compatible with aquatic product matrices has become an urgent technical problem to be solved in the field of food testing. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the quantitative determination of total phosphate in aquatic products using acid hydrolysis combined with liquid chromatography-tandem mass spectrometry. This method first completely extracts various phosphates from aquatic products, then converts them into orthophosphates through high-temperature acid hydrolysis. The orthophosphate content is then accurately determined using liquid chromatography-tandem mass spectrometry, thereby achieving accurate determination of total phosphate in aquatic products. This provides a new technical means for the detection of polyphosphates in aquatic products and reliable technical support for the routine monitoring and detection of phosphate water-retaining agents in aquatic product processing.

[0007] To achieve the above objectives, the present invention provides a method for the quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry, comprising the following steps: S1. After homogenizing the sample to be tested, add ultrapure water, vortex, sonicate, and centrifuge to obtain the extract; mix the extract with an acid solution for acid hydrolysis. After hydrolysis, adjust the pH of the hydrolysate to 3-7, make up to volume, and centrifuge to obtain the supernatant; mix the supernatant with the orthophosphate isotope internal standard solution to obtain the test solution. S2. Dilute the orthophosphate standard into eight orthophosphate standard solutions of different concentrations, and add orthophosphate isotope internal standard solution to the above orthophosphate standard solutions of different concentrations respectively to obtain a series of standard solutions to be tested. S3. Inject the standard series solutions to be tested into the liquid chromatography-tandem mass spectrometer, adjust the chromatographic and mass spectrometric conditions, and obtain the standard curve; then inject the test solution into the liquid chromatography-tandem mass spectrometer for detection to obtain the total phosphate content in aquatic products. The chromatographic column used in S3 was an Acclaim Trinity Q1 column with dimensions of 3.0 mm × 100 mm and a diameter of 3 μm. This column can achieve ideal separation of orthophosphate, with suitable peak retention time and symmetrical peak shape. The mobile phase was an ammonium formate solution with a concentration of 12.5-50 mmol / L and a pH of 3.0-4.5. In S3, the mass spectrometry conditions selected were: electrospray ionization source in negative ion mode, and the scanning mode was: select reaction monitoring mode.

[0008] In this invention, the mass-to-volume ratio of the homogenized sample to ultrapure water in S1 is 1 g: 20 mL; the vortexing time is 1 min; the sonication time is 30 min; the centrifugation speed is 12000 r / min, and the centrifugation time is 5 min; the volume ratio of the extract to the acid solution is 1:0.05-0.2, and the volume fraction of the acid solution is 30%, including sulfuric acid solution, hydrochloric acid solution, or nitric acid solution. In this invention, the acid hydrolysis temperature in S1 is 70-90℃, and the time is 40-80 min.

[0009] In this invention, after hydrolysis is completed in step S1, 10 mL of ultrapure water is added to the hydrolysis system, and then the pH of the hydrolysate is adjusted to 3-7. The method for adjusting the pH of the hydrolysate to 3-7 is as follows: a 1 mol / L NaOH aqueous solution is added to the hydrolysate until the pH of the hydrolysate reaches 3-7.

[0010] In this invention, the volume ratio of the supernatant to the orthophosphate isotope internal standard solution in S1 is 1:0.02, and the orthophosphate isotope internal standard (P... 18 O4 3- The concentration of the solution used is 100 mg / L.

[0011] In this invention, the concentrations of orthophosphate in the series of standard solutions to be tested in S2 are 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, and 20.0 mg / L, respectively, and the concentration of the orthophosphate isotope internal standard solution is 2.0 mg / L.

[0012] In this invention, under S3 chromatographic conditions, the flow rate is 0.4 mL / min and the injection volume is 10 μL.

[0013] In this invention, the S3 mass spectrometry conditions are as follows: spray voltage is 2.5 kV, vaporization chamber temperature is 300 °C, ion transport capillary temperature is 325 °C; sheath gas pressure is 50 arb, and sheath gas is nitrogen; auxiliary gas pressure is 20 arb, and auxiliary gas is nitrogen.

[0014] In this invention, the mass spectrometry detection parameters in S3 are as follows: the precursor ion of orthophosphate is 97.0, the quantitative daughter ion is 79.0, the qualitative daughter ion is 63.0, the collision energies are 18 eV and 50 eV respectively, and the S-lens voltage is 44 V; 180 4- The precursor ion of the orthophosphate isotope internal standard is 105.0; the product ion is 85.0; the collision energy is 18 eV; and the S-lens voltage is 44 V.

[0015] The present invention has the following beneficial effects: The present invention provides a method for the quantitative detection of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry. By optimizing and setting reasonable ranges for the parameters of the entire process of sample pretreatment, acid hydrolysis, chromatographic separation and mass spectrometry detection, this method effectively solves the technical defects of existing methods for detecting phosphate in aquatic products, such as insufficient sensitivity, large quantitative deviation, weak resistance to matrix interference and cumbersome operation. It has the advantages of detection accuracy, convenient operation and practical application adaptability. It shows significant advantages in detection performance, operability and regulatory adaptability, and provides a reliable technical means for the routine quantitative detection of total phosphate in aquatic products and food safety supervision.

[0016] This invention optimizes the range of parameters such as reagents, temperature, and time in acid hydrolysis, enabling the complete hydrolysis of polyphosphates such as pyrophosphate, tripolyphosphate, trimetaphosphate, and hexametaphosphate. This effectively avoids the quantitative deviation problems caused by incomplete hydrolysis of polyphosphates and degradation during extraction in existing methods.

[0017] This invention utilizes the Acclaim Trinity Q1 chromatographic column, which features multiple retention modes, to effectively separate orthophosphate from impurities such as proteins, inorganic salts, and small organic molecules in aquatic product matrices. Combined with isotope internal standard quantification, the matrix effect value for typical aquatic product matrices such as shrimp and fish is 94.3%-96.1%, with virtually no matrix interference. It eliminates the need for complex purification methods such as solid-phase extraction, making it suitable for detecting different aquatic product matrices and solving the technical problems of existing methods being susceptible to matrix influence and having poor quantitative accuracy.

[0018] This invention uses ultrapure water as the extraction solvent, and acid hydrolysis can be completed with just a conventional constant temperature water bath. The entire process involves no toxic reducing agents or complex consumables, and the parameter ranges of each operation step are compatible with conventional laboratory operating conditions, which greatly reduces the difficulty of detection operations and the cost of consumables. At the same time, it simplifies the pretreatment process, avoids cumbersome purification steps, is suitable for rapid pretreatment of batch samples, and further improves the overall detection efficiency.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is a secondary mass spectrometry scan of the orthophosphate and its isotope internal standard of the present invention; in, Figure 1 In the image, 'a' represents the secondary mass spectrometry scan of orthophosphate. Figure 1 b in 18 Secondary mass spectrometry scan of O4-orthophosphate isotope internal standard; Figure 2 This is a graph showing the effect of the concentration of ammonium formate solution on the chromatographic behavior of orthophosphate, as tested in this invention. Figure 3 This is a graph showing the effect of pH of ammonium formate solution on the chromatographic behavior of orthophosphate, as tested in this invention. Figure 4 This is a diagram showing the experimental results of the types of acids in this invention; Figure 5 This is a graph showing the experimental results of the amount of acid solution used in this invention; Figure 6 This is a graph showing the experimental results of the acid hydrolysis temperature of this invention; Figure 7 This is a graph showing the experimental results of acid hydrolysis time in this invention; Figure 8 This is the standard curve constructed in this invention; Figure 9 This is an SRM chromatogram of the orthophosphate and its isotope internal standard of the present invention; in, Figure 9 In this context, 'a' represents the standard solution. Figure 9 b in the text represents the blank solution; Figure 9 c in the text represents the shrimp sample solution. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0022] Instruments and Reagents: Triple quadrupole mass spectrometer: Thermo TSQ Vantage (USA); High-speed refrigerated centrifuge: Beckman JNX-30 (USA); Ultrapure water treatment system: Merck Milli-Q (18.2 MΩ) (Germany). Formic acid (CNW, LC-MS grade); Ammonium formate (CNW, LC-MS grade); Acetonitrile (Merck, chromatographic grade); Nitric acid (Chinese medicine, analytical grade), Hydrochloric acid (Chinese medicine, analytical grade), Sulfuric acid (Chinese medicine, analytical grade), Sodium hydroxide (NaOH, Chinese medicine, analytical grade); Ultrapure water for the experiment was prepared by the Milli-Q ultrapure water system. Orthophosphate (GBW(E)083180, 1000 mg / L), pyrophosphate (BW20316-1000-50, 1000 mg / L), tripolyphosphate (BW20319-1000-50, 1000 mg / L), trimetaphosphate (BW20318-1000-50, 1000 mg / L), and hexametaphosphate (BW20315-1000, 1000 mg / L) standards were purchased from Tanmo Quality Inspection Technology; orthophosphate isotope internal standard (P 18 O4 3- The fish meal (IR-30936, 96%) was purchased from Shanghai Zhenzhun Biotechnology; the phosphate control sample in the fish meal (BY400138, 14.3 g / kg) was purchased from Beijing Zhenxiang Technology. The experimental water was purchased from local farmers' markets and supermarkets in Putuo District, Zhoushan City.

[0023] Mass spectrometry condition optimization: A 5.0 mg / L orthophosphate standard solution was prepared and injected into the mass spectrometer. Precursor ion scanning was performed in negative ion mode. The results showed that the main molecular ion peak was at a mass-to-charge ratio (m / z) of 97.0, and the corresponding detectable ion was dihydrogen phosphate (H₂PO₄). - No hydrogen phosphate (HPO4) with an m / z of 48.0 was found. 2- The mass spectrum peak and the phosphate (PO4) at m / z 31.7 3- The mass spectrometry peaks were observed. A secondary fragment ion scan was performed on m / z 97.0 as the parent ion, revealing m / z 79.1 and m / z 63.0 as the two main daughter ions, corresponding to metaphosphate (PO3) as the detected ions. - ) and metaphosphate (PO2) - Based on the relevant results, the following pathway can be inferred for the intrasource cleavage of orthophosphate: first, a water molecule is removed from the dihydrogen phosphate ion to form a metaphosphate ion; then, the metaphosphate ion removes an oxygen atom to form a metaphosphite ion. Figure 1As can be seen, the scanning ion map of the orthophosphate isotope internal standard shows that the intraspectral fragmentation path of the isotope is consistent with that of the target analyte, orthophosphate. Based on the selected parent and daughter ions, mass spectrometry parameters such as lens voltage and collision energy were optimized. The optimized mass spectrometry detection parameters are shown in Table 1.

[0024] Table 1 Optimized mass spectrometry detection parameters

[0025] Note: a: (Quantitative ion).

[0026] Optimization of chromatographic conditions: Selection of chromatographic column: Six chromatographic columns were selected for the separation of orthophosphate: Hypersil Gold C18 (2.1 mm × 100 mm, 1.9 μm), Synergi Max-RP (2.0 mm × 100 mm, 2.5 μm), Acquity HSS T3 (2.1 mm × 100 mm, 1.8 μm), Acquity BEH Amide (3.0 mm × 150 mm, 1.7 μm), Acclaim Trinity P1 (2.1 mm × 100 mm, 3 μm), and Acclaim Trinity Q1 (3.0 mm × 100 mm, 3 μm).

[0027] Orthophosphate is a highly polar compound, exhibiting very weak retention (less than 1.6 min) on three columns: Hypersil Gold C18, Synergi Max-RP, and Acquity HSS T3, with peak tailing. Separation using a hydrophilic Acquity BEH Amide column failed to detect the target analyte. Finally, this invention selected two columns with multiple retention modes (Acclaim Trinity P1 column with strong cation exchange, weak anion exchange, and reversed-phase retention modes; and Acclaim Trinity Q1 column with weak cation exchange, weak anion exchange, and reversed-phase retention modes) for target analyte separation. The results showed that, compared to the Acclaim Trinity Q1 column, the Acclaim Trinity P1 column exhibited relatively weaker retention of the target analyte and peak tailing. The Acclaim Trinity Q1 column achieved satisfactory separation of orthophosphate, with suitable peak retention time and symmetrical peak shape.

[0028] Mobile phase optimization: Using the same detection procedure as in Example 1, the effect of ammonium formate solution concentrations of 12.5 mmol / L, 25 mmol / L, 37.5 mmol / L, and 50 mmol / L in the mobile phase on the orthophosphate chromatographic behavior was tested. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that changes in ammonium formate concentration significantly altered the retention characteristics of the target analyte. At low concentrations (12.5 mmol / L), orthophosphate exhibited strong chromatographic retention and significant peak broadening. As the ammonium formate concentration increased, the peak shape gradually improved, symmetry increased, peak width narrowed, and elution time shortened accordingly. When the ammonium formate concentration reached 50 mmol / L, the orthophosphate peak was sharp and symmetrical, with a retention time of less than 4 min.

[0029] Using the same detection procedure as in Example 1, the effect of the pH of the ammonium formate solution at 3.0, 3.5, 4.0, and 4.5 on the orthophosphate chromatographic behavior was tested. The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that as the pH of the mobile phase decreases, the elution time of the target analyte on the chromatographic column shifts slightly forward, but the change is small. The effect of pH on the chromatographic peak response is more significant; as the pH decreases, the peak response gradually strengthens. At a pH of 3.0, the chromatographic peak shape of orthophosphate is optimal, and the response value is the highest.

[0030] Optimization of acid hydrolysis conditions: Common polyphosphates used in aquatic product processing (pyrophosphate, tripolyphosphate, trimetaphosphate, hexametaphosphate, etc.) can be converted into orthophosphate under acidic conditions. This invention prepared single-standard solutions of four polyphosphates at a concentration of 4 mg / L and conducted acid hydrolysis experiments. The acid hydrolysis recovery rate was defined as the percentage of orthophosphate concentration obtained after acid hydrolysis compared to the theoretically complete conversion of the polyphosphate to orthophosphate.

[0031] Acid type experiment: First, three commonly used laboratory acids—sulfuric acid, hydrochloric acid, and nitric acid—were selected, and 30% (v / v) acid solutions were prepared for each. This was used to investigate the effect of acid type on the acid hydrolysis efficiency of four polyphosphates. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that all three acids have good acid hydrolysis efficiency for the four target substances, and all can meet the experimental requirements.

[0032] Experiment on the amount of acid used: Volumes of 30% nitric acid solution (10 μL, 20 μL, 50 μL, 100 μL, 200 μL, and 400 μL) were selected to investigate the effect of the amount of acid solution used on the acid hydrolysis efficiency of the target compound. The results are as follows: Figure 5 As shown. From Figure 5 It can be seen that as the amount of acid solution used increases, the acid hydrolysis efficiency of the four polyphosphates gradually increases. When the volume reaches 200 μL, the acid hydrolysis efficiency reaches its highest level. Further increasing the amount of acid has no significant effect on improving the conversion efficiency of the target analyte.

[0033] Acid hydrolysis temperature experiment: The effects of water bath conditions at 25℃, 50℃, 70℃, and 90℃ on the acid hydrolysis efficiency of the target compound were investigated, and the results are as follows: Figure 6 As shown. From Figure 6 It can be seen that as the acid hydrolysis temperature increases, the conversion efficiency of the four polyphosphates also increases. When the temperature reaches 90℃, the acid hydrolysis efficiency reaches a relatively high level.

[0034] Acid hydrolysis time experiment: The effects of acid hydrolysis times of 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, and 80 min on the acid hydrolysis efficiency of four target compounds were investigated, and the results are as follows: Figure 7 As shown. From Figure 7 It can be seen that the acid hydrolysis efficiency of the target substance gradually increases with the increase of heating time, and reaches a peak when the heating time is 60 min. Further increasing the heating time does not significantly improve the conversion efficiency of the target substance.

[0035] Extraction solvent optimization: The effects of ultrapure water and NaOH solutions of different concentrations as extraction solvents on the extraction recovery rate of phosphate in aquatic products were investigated. The results showed that the extraction recoveries of orthophosphate in aquatic products by ultrapure water, 10 mmol / L NaOH solution, 20 mmol / L NaOH solution, 50 mmol / L NaOH solution, and 100 mmol / L NaOH solution were 94.9%, 90.3%, 87.8%, 74.5%, and 84.2%, respectively. These results indicate that ultrapure water has a relatively high extraction recovery rate for orthophosphate. To further investigate the effect of ultrapure water on the extraction recovery rate of four polyphosphates, four samples were selected, and appropriate amounts of pyrophosphate, tripolyphosphate, trimetaphosphate, and hexametaphosphate standard solutions were added to prepare individually spiked samples of the four polyphosphates. The recoveries of the spiked samples of the four polyphosphates ranged from 93.2% to 101.9%, which met the experimental requirements. Therefore, ultrapure water was chosen as the extraction solvent for the detection of total phosphate in aquatic products.

[0036] Matrix effect, working curve and detection limit: A standard curve was plotted with the concentration of orthophosphate on the x-axis and the ratio of the peak area of ​​the target analyte to that of the internal standard on the y-axis. A good linear relationship was observed in the concentration range of 0.1–20.0 mg / L, with the linear equation y = 0.023047 + 1.01045x and a correlation coefficient of 0.9991. The results are shown in [Figure number missing]. Figure 8 This study evaluated the matrix effect (ME) by comparing the slope of the standard curve prepared from the matrix solution obtained after sample pretreatment with the slope of the standard curve prepared from the pure solvent. The results showed that the ME values ​​for shrimp and fish matrices were 96.1% and 94.3%, respectively, indicating virtually no matrix effect. Under the experimental conditions, based on peak response values ​​at 3x and 10x noise levels, the method detection limits for phosphate in both shrimp and fish matrices were 0.03 g / kg, and the quantitation limits were both 0.10 g / kg.

[0037] GB 2760-2024, the standard for the use of food additives, stipulates that the maximum permitted amount of phosphate in frozen aquatic products is 5 g / kg. Compared with this standard limit, the detection sensitivity and linear range of this method both meet the detection requirements, demonstrating good practical application value.

[0038] Example 1 S1. Take 1.0 g of homogenized and frozen shrimp meat, add orthophosphate at a spiking concentration of 1.0 g / kg, then add 20 mL of ultrapure water, vortex for 1 min, sonicate for 30 min, and centrifuge at 12000 r / min for 5 min to obtain the extract; transfer 1.0 mL of the extract to a 50 mL graduated centrifuge tube, add 200 μL of 30% nitric acid solution, and hydrolyze in a 90℃ water bath for 60 min. After hydrolysis, add 10 mL of ultrapure water, adjust the pH to 7 with 1 mol / L NaOH aqueous solution, and finally bring the volume to 50 mL with ultrapure water; centrifuge at 3000 r / min for 5 min to obtain the supernatant; transfer 1.0 mL of the supernatant to a sample bottle, add 20 μL of 100 mg / L orthophosphate isotope internal standard (P 18 O4 3- The solution used is used to obtain the solution to be tested; S2. Dilute the orthophosphate standard with ultrapure water to obtain a stock solution with a concentration of 100.0 mg / L. Then, dilute the stock solution with ultrapure water to obtain orthophosphate standard solutions of different concentrations. Add orthophosphate isotope internal standard solution to the above orthophosphate standard solutions of different concentrations to obtain a series of standard solutions to be tested. In the series of standard solutions to be tested, the concentrations of orthophosphate were 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, and 20.0 mg / L, respectively, and the concentration of the orthophosphate isotope internal standard solution was 2.0 mg / L. S3. Inject the standard series solutions to be tested into the liquid chromatography-tandem mass spectrometer, adjust the chromatographic and mass spectrometric conditions, and obtain the standard curve with the concentration of orthophosphate as the abscissa and the ratio of the peak area of ​​the target analyte to the peak area of ​​the internal standard as the ordinate. Then, inject the test solution into the liquid chromatography-tandem mass spectrometer for detection to obtain the total phosphate content in aquatic products. Chromatographic conditions: Column: Acclaim Trinity Q1 column (3.0 mm × 100 mm, 3 μm), mobile phase: 50 mmol / L ammonium formate solution adjusted to pH 3.0 with formic acid, flow rate: 0.4 mL / min, injection volume: 10 μL.

[0039] Mass spectrometry conditions: Ion source: Electrospray ionization source, negative ion mode (ESI) - The scanning method was: Selected Reaction Monitoring (SRM); Spray voltage: 2.5 kV; Vaporization chamber temperature: 300 °C; Ion transport capillary temperature: 325 °C; Sheath gas (nitrogen): 50 alb; Auxiliary gas (nitrogen): 20 alb; Collision gas: argon. The mass spectrometry detection parameters were the same as those in Table 1 above.

[0040] Example 2 The method is basically the same as that provided in Example 1, except that in S1, orthophosphate is added at a spiked concentration of 5.0 g / kg.

[0041] Example 3 The method is basically the same as that provided in Example 1, except that in S1, orthophosphate is added at a spiked concentration of 10 g / kg.

[0042] Example 4 The method is basically the same as that provided in Example 1, except that the sample in S1 is fish meat.

[0043] Example 5 The method is basically the same as that provided in Example 1, except that the sample in S1 is fish meat and orthophosphate is added at a spiked concentration of 5.0 g / kg.

[0044] Example 6 The method is basically the same as that provided in Example 1, except that the sample in S1 is fish meat and orthophosphate is added at a spiked concentration of 10 g / kg.

[0045] Detection: For each group of samples in Examples 1-6, 6 parallel experiments were conducted to determine the intraday recovery rate and intraday relative standard deviation (RSD); the interday recovery rate and interday precision were determined after 3 consecutive days of testing. The results are shown in Table 2.

[0046] Table 2 Recovery and precision of the method

[0047] As shown in Table 2, the intra-day recoveries of total phosphate in shrimp and fish were 89.8%-102.8% and 88.7%-105.8%, respectively; the inter-day recoveries were 90.9%-106.4% and 90.2%-105.8%, respectively, and the intra-day and inter-day RSDs of the detection methods were ≤10.0% and 9.6%, respectively.

[0048] Example 7 S1. Take 1.0 g of homogenized certified phosphate standard sample (total phosphate in fish meal, BY400138), add 20 mL of ultrapure water, vortex for 1 min, sonicate for 30 min, centrifuge at 12000 r / min for 5 min to obtain the extract; transfer 1.0 mL of the extract to a 50 mL graduated centrifuge tube, add 200 μL of 30% nitric acid solution, and hydrolyze in a 90℃ water bath for 60 min. After hydrolysis, add 10 mL of ultrapure water, adjust the pH to 7 with 1 mol / L NaOH aqueous solution, and finally bring the volume to 50 mL with ultrapure water; centrifuge at 3000 r / min for 5 min to obtain the supernatant; transfer 1.0 mL of the supernatant to a sample bottle, add 20 μL of 100 mg / L orthophosphate isotope internal standard (P 18 O4 3- The solution used is used to obtain the solution to be tested; S2. Dilute the orthophosphate standard with ultrapure water to obtain a stock solution with a concentration of 100.0 mg / L. Then, dilute the stock solution with ultrapure water to obtain orthophosphate standard solutions of different concentrations. Add orthophosphate isotope internal standard solution to the above orthophosphate standard solutions of different concentrations to obtain a series of standard solutions to be tested. In the series of standard solutions to be tested, the concentrations of orthophosphate were 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, and 20.0 mg / L, respectively, and the concentration of the orthophosphate isotope internal standard solution was 2.0 mg / L. S3. Inject the standard series solutions to be tested into the liquid chromatography-tandem mass spectrometer, adjust the chromatographic and mass spectrometric conditions, and obtain the standard curve with the concentration of orthophosphate as the abscissa and the ratio of the peak area of ​​the target analyte to the peak area of ​​the internal standard as the ordinate. Then, inject the test solution into the liquid chromatography-tandem mass spectrometer for detection to obtain the total phosphate content in aquatic products. Chromatographic conditions: Column: Acclaim Trinity Q1 column (3.0 mm × 100 mm, 3 μm), mobile phase: 50 mmol / L ammonium formate solution adjusted to pH 3.0 with formic acid, flow rate: 0.4 mL / min, injection volume: 10 μL.

[0049] Mass spectrometry conditions: Ion source: Electrospray ionization source, negative ion mode (ESI) - The scanning method was: Selected Reaction Monitoring (SRM); Spray voltage: 2.5 kV; Vaporization chamber temperature: 300 °C; Ion transport capillary temperature: 325 °C; Sheath gas (nitrogen): 50 alb; Auxiliary gas (nitrogen): 20 alb; Collision gas: argon. The mass spectrometry detection parameters were the same as those in Table 1 above.

[0050] The test results in Example 7 showed that the total phosphate content in the fish meal was 16.7 ± 0.2 g / kg. This is within an acceptable range compared to the expected concentration of total phosphate (11.6 g / kg - 17.0 g / kg) on ​​the certificate.

[0051] Example 8 S1. Take 1.0 g of homogenized and frozen shrimp meat, then add 20 mL of ultrapure water, vortex for 1 min, sonicate for 30 min, and centrifuge at 12000 r / min for 5 min to obtain the extract; transfer 1.0 mL of the extract to a 50 mL graduated centrifuge tube, add 200 μL of 30% nitric acid solution, and hydrolyze in a 90℃ water bath for 60 min. After hydrolysis, add 10 mL of ultrapure water, adjust the pH to 7 with 1 mol / L NaOH aqueous solution, and finally make up to 50 mL with ultrapure water; centrifuge at 3000 r / min for 5 min to obtain the supernatant; transfer 1.0 mL of the supernatant to a sample bottle, add 20 μL of 100 mg / L orthophosphate isotope internal standard (P 18 O4 3- The solution used is used to obtain the solution to be tested; S2. Dilute the orthophosphate standard with ultrapure water to obtain a stock solution with a concentration of 100.0 mg / L. Then, dilute the stock solution with ultrapure water to obtain orthophosphate standard solutions of different concentrations. Add orthophosphate isotope internal standard solution to the above orthophosphate standard solutions of different concentrations to obtain a series of standard solutions to be tested. In the series of standard solutions to be tested, the concentrations of orthophosphate were 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, and 20.0 mg / L, respectively, and the concentration of the orthophosphate isotope internal standard solution was 2.0 mg / L. Ultrapure water was used as the blank solution. S3. Inject the standard series solutions and blank solutions to be tested into the liquid chromatography-tandem mass spectrometer, adjust the chromatographic and mass spectrometric conditions, and obtain the standard curve with the concentration of orthophosphate as the abscissa and the ratio of the peak area of ​​the target analyte to the peak area of ​​the internal standard as the ordinate. Then, inject the test solution into the liquid chromatography-tandem mass spectrometer for detection to obtain the total phosphate content in aquatic products. Chromatographic conditions: Column: Acclaim Trinity Q1 column (3.0 mm × 100 mm, 3 μm), mobile phase: 50 mmol / L ammonium formate solution adjusted to pH 3.0 with formic acid, flow rate: 0.4 mL / min, injection volume: 10 μL.

[0052] Mass spectrometry conditions: Ion source: Electrospray ionization source, negative ion mode (ESI) - The scanning method was: Selected Reaction Monitoring (SRM); Spray voltage: 2.5 kV; Vaporization chamber temperature: 300 °C; Ion transport capillary temperature: 325 °C; Sheath gas (nitrogen): 50 alb; Auxiliary gas (nitrogen): 20 alb; Collision gas: argon. The mass spectrometry detection parameters were the same as those in Table 1 above.

[0053] See results Figure 9 ,from Figure 9 It can be seen that under the chromatographic and mass spectrometric conditions set in this invention, orthophosphate elutes at 3.32 min. 18 O 4- The orthophosphate isotope internal standard eluted at 3.31 min, with retention times close and non-overlapping, indicating good chromatographic separation. This allows for independent and accurate detection of both the target analyte and the internal standard, providing a clear characteristic retention time reference for subsequent quantitative calculations. The process blank solution showed no obvious chromatographic peaks within the characteristic retention time range of orthophosphate and the isotope internal standard, exhibiting only baseline noise. This indicates that the reagents, sample pretreatment process, and instrument detection system used in this invention are free from background contamination and will not cause false positive interference in the detection of the target analyte, fully demonstrating the excellent specificity of this method. The elution times of the shrimp sample solution in the quantitative, qualitative, and internal standard ion channels were completely consistent with the corresponding elution times of the standard solution, and the chromatographic peaks were clear without obvious tailing or broadening. This indicates that components such as proteins and fats in the aquatic product matrix did not significantly interfere with the chromatographic separation and mass spectrometry detection of orthophosphate. This invention's method can effectively detect the target analyte from actual aquatic product matrices and has good applicability to actual sample detection.

[0054] Ten samples of frozen shrimp and shrimp meat sold in Putuo District, Zhoushan City in 2025 were tested according to the method in Example 7. The test results showed that the total phosphate content in the samples was 2.45-5.12 g / kg, and one frozen shrimp sample exceeded the limit value.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for quantitative determination of total phosphate in aquatic products using acid hydrolysis combined with liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: S1. After homogenizing the sample to be tested, add ultrapure water, vortex, sonicate, and centrifuge to obtain the extract; The extract was mixed with an acid solution for acid hydrolysis. After hydrolysis, the pH of the hydrolysate was adjusted to 3-7, and the solution was brought to a final volume and centrifuged to obtain the supernatant. The supernatant was then mixed with the orthophosphate isotope internal standard solution to obtain the test solution. S2. Dilute the orthophosphate standard into eight orthophosphate standard solutions of different concentrations, and add orthophosphate isotope internal standard solution to the above orthophosphate standard solutions of different concentrations respectively to obtain a series of standard solutions to be tested. S3. Inject the standard series solutions to be tested into the liquid chromatography-tandem mass spectrometer, adjust the chromatographic and mass spectrometric conditions, and obtain the standard curve; then inject the test solution into the liquid chromatography-tandem mass spectrometer for detection to obtain the total phosphate content in aquatic products. The chromatographic conditions used in S3 were: an Acclaim Trinity Q1 column and an ammonium formate solution with a concentration of 12.5-50 mmol / L and a pH of 3.0-4.

5. In S3, the mass spectrometry conditions selected were: electrospray ionization source in negative ion mode, and the scanning mode was: select reaction monitoring mode.

2. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The mass-to-volume ratio of the homogenized sample to ultrapure water in S1 is 1g:20mL. The volume ratio of the extract to the acid solution is 1:0.05-0.2, and the volume fraction of the acid solution is 30%, including sulfuric acid solution, hydrochloric acid solution or nitric acid solution.

3. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The acid hydrolysis temperature in S1 is 70-90℃, and the time is 40-80 min.

4. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The volume ratio of the supernatant to the orthophosphate isotope internal standard solution in S1 is 1:0.02, and the concentration of the orthophosphate isotope internal standard solution is 100 mg / L.

5. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In the series of standard solutions to be tested in S2, the concentrations of orthophosphate were 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, and 20.0 mg / L, respectively, and the concentration of the orthophosphate isotope internal standard solution was 2.0 mg / L.

6. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, Under S3 chromatographic conditions, the flow rate was 0.4 mL / min and the injection volume was 10 μL.

7. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, In the S3 mass spectrometry conditions, the spray voltage was 2.5 kV, the vaporization chamber temperature was 300 °C, and the ion transport capillary temperature was 325 °C; the sheath gas pressure was 50 arb, and the auxiliary gas pressure was 20 arb.

8. The method for quantitative determination of total phosphate in aquatic products by acid hydrolysis combined with liquid chromatography-tandem mass spectrometry according to claim 1 or 7, characterized in that, The mass spectrometry detection parameters for S3 detection are as follows: the precursor ion of orthophosphate is 97.0, the quantitative daughter ion is 79.0, the qualitative daughter ion is 63.0, the collision energies are 18 eV and 50 eV, respectively, and the S-lens voltage is 44 V. 18 The precursor ion of the O4-orthophosphate isotope internal standard is 105.0; the product ion is 85.0, the collision energy is 18 eV, and the S-lens voltage is 44 V.

Images